Intracellular Trafficking Governs the Processing of the Amyloid Precursor Protein and the Secretion of Beta-Amyloid

One of the hallmarks of Alzheimer’s disease (AD) is the pathological accumulation of β-amyloid (Aβ) in the brains of AD patients. Oligomeric and fibrillar aggregates of Aβ have been shown to be neurotoxic to neurons and hippocampal slices. Therefore, limiting Aβ production is an important area of research in order to delay or stop AD progression. Aβ is produced by amyloidogenic cleavage of amyloid precursor protein (APP). Amyloidogenic cleavage requires ectodomain removal by β-secretase and intramembrane γ-cleavage by γ-secretase to release Aβ products ranging from 38-43 residues. Work from our lab has shown that APP and γ-secretase are resident proteins of the lysosome. Furthermore, the acidic environment of lysosomes that promotes the aggregation of Aβ. While many lines of evidence demonstrate that APP internalization is important to the Aβ production, the intracellular itinerary of APP, from production to cleavage, is unclear. In order to follow the intracellular trafficking of APP and Aβ, we have applied various microscopy techniques, in combination with fluorescently-tagged proteins. Using a photoactivatable mutant of GFP (paGFP), we accurately photoactivated nascent APP and followed its trafficking to lysosomes. To our surprise, we found that APP was delivered to lysosomes, where it is cleaved by γ-secretase, through an entirely intracellular pathway. This intracellular pathway was dependent upon an interaction between APP and adaptor protein 3. We found that the interaction between APP and AP-3 is dependent on the 709YTSI712 tyrosine motif. Furthermore, phosphorylation of the serine within this motif, by PKCε, can disrupt this interaction. By decreasing APP trafficking to lysosomes, through disrupting the APP/AP-3 interaction we decreased the production of Aβ. While lysosomes have traditionally been thought to be responsible for cellular waste disposal, they also have a secretory role in a number of cell types; including neurons. We demonstrate that lysosomes are not only responsible for the production of Aβ, but may also be responsible for the secretion of lysosomal Aβ into the extracellular space. This research may provide new therapeutic targets to limit the production and release of Aβ

A Mathematical Model of Murine Metabolic Regulation by Leptin: Energy Balance and Defense of a Stable Body Weight

We have developed a physiologically based mathematical model, with parameters derived from published experimental data, to simulate the regulatory effects of the leptin pathway on murine energy homeostasis. Model outcomes are consistent with data reported in the literature and reproduce key characteristics of the energy regulatory system, including compensatory responses that counteract changes in body weight and the failure of this ability when the leptin pathway is disrupted. Our model revealed the possibility of multiple steady states for body weight. It also provided a unified theoretical framework for two historically antagonistic hypotheses regarding body weight regulation (“set-point” versus “settling point”). Finally, our model has identified potential avenues for future investigations.National Institutes of Health (U.S.) (grant NIH CA80124)National Institutes of Health (U.S.) (grant NIH CA85140)National Institutes of Health (U.S.) (grant NIH CA96915)National Institutes of Health (U.S.) (grant NIH CA115767

Adipogenesis and angiogenesis : roles in tissue engineering and glucose metabolism

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009.Cataloged from PDF version of thesis.Includes bibliographical references (p. 118-127).Adipose tissue serves two main functions in the body: (1) it is the body's primary energy depot; and (2) it also serves as an important endocrine organ, producing and secreting various enzymes, growth factors, cytokines, and hormones. Both of these functions require ample access to circulating blood. Many aspects of angiogenesis during adipose tissue expansion remain poorly understood. Adipocytes produce a large variety of molecules involved in angiogenesis, and obesity is associated with elevated circulating levels of Vascular Endothelial Growth Factor (VEGF). Our lab has previously shown that angiogenesis and adipogenesis are mutually dependent via a VEGF receptor 2 (VEGFR2)-mediated mechanism. Since then several other studies have reinforced a role for the VEGF-VEGFR system in energy metabolism. For example, genetically obese mice treated with anti-VEGF antibody had lower fat pad weights, but the VEGF receptor responsible for this observation is not known. There is also disagreement on the cell type(s) responsible for fat tissue's angiogenic capability, with some studies supporting a dominant role for adipocytes, while others attribute most of the angiogenic capacity to the adipose tissue stromal cells (ASC). This thesis project aimed to fill some of these gaps by examining the angiogenic capacity of adipose tissue relative to other tissues, the effects of VEGFR-1 and R-2 blockade in mouse models of adipogenesis and diet-induced obesity, the respective angiogenic capabilities of adipocytes and ASC, and the possibility of harnessing the angiogenic potential of adipose tissue for vascular tissue engineering.(cont.) In addition, a physiologically-based mathematical model was developed to simulate the regulatory effects of the leptin pathway on murine energy homeostasis.by Joshua Tam.Ph.D

CSE: U: Mixed-initiative Personal Assistant Agents

Specification and implementation of flexible human-computer dialogs is challenging because of the complexity involved in rendering the dialog responsive to a vast number of varied paths through which users might desire to complete the dialog. To address this problem, we developed a toolkit for modeling and implementing task-based, mixed-initiative dialogs based on metaphors from lambda calculus. Our toolkit can automatically operationalize a dialog that involves multiple prompts and/or sub-dialogs, given a high-level dialog specification of it. The use of natural language with the resulting dialogs makes the flexibility in communicating user utterances commensurate with that in dialog completion paths—an aspect missing from commercial assistants like Siri. Our results demonstrate that the dialogs authored with our toolkit support the end user’s completion of a human-computer dialog in a manner that is most natural to them—in a mixed-initiative fashion—that resembles human-human interaction

The Effects of Chronic Sleep Deprivation on Tumor Necrosis Factor Alpha and Bone Strength in Menopausal Rats

Osteoporosis affects one in four post-menopausal women, and approximately one in three of those women will experience a fracture due to osteoporosis in their lifetime. Sleep deprivation may be a contributing factor in the acceleration of osteoporosis due to the increase in pro-inflammatory cytokine production which may potentiate the diminished estrogen signaling. Tumor-necrosis factor alpha-type (TNFα), a pro-inflammatory cytokine of interest, stimulates osteoprotegerin ligand (OPGL) to increase the degradation of bone. Bisphosphonates, like Zolendronate, are the most commonly prescribed treatment for osteoporosis because they decrease osteoclastic activity. This study analyzed the effects of chronic sleep deprivation and Zolendronate on TNFα concentration and relative bone strength of thirty-two ovariectomized Wistar rats in a five-week protocol; groups included control (C), sleep deprived (SD), sleep deprived with Zolendronate (SDZ), and Zolendronate (Z). TNFα concentrations were determined by enzyme-linked immunoassay and bone strength was determined by a three-point bending test. The calculated TNFα concentration values for the groups were: C (M=23.97 pg/mL, =1.87 pg/mL), SD (M=25.56 pg/mL, =2.56 pg/mL), SDZ (M=24.02 pg/mL, =2.17 pg/mL), and Z (M=27.26 pg/mL, =2.22 pg/mL). The calculated bone strength values for the groups followed: C (M=161.42 N, =19.37 N), SD (M=162.21 N, =24.29 N), SDZ (M=165.97 N, =22.61 N), and Z (M=156.44 N, =20.46 N). There were significant differences (p \u3c .01) in serum concentration of TNFα in the group taking Zolendronate, but we found no differences in bone strength between the groups – but the trends suggest that in larger sample sizes, differences would emerge. We believe that further research measuring the changes in cytokine concentration throughout a longer sleep deprivation protocol would be a fruitful

Blockade of VEGFR2 and Not VEGFR1 Can Limit Diet-Induced Fat Tissue Expansion: Role of Local versus Bone Marrow-Derived Endothelial Cells

Background: We investigated if new vessel formation in fat involves the contribution of local tissue-derived endothelial cells (i.e., angiogenesis) or bone marrow-derived cells (BMDCs, i.e. vasculogenesis) and if antiangiogenic treatment by blockade of vascular endothelial growth factor (VEGF) receptors can prevent diet-induced obesity (DIO). Methodology/Principal Findings: We performed restorative bone marrow transplantation into wild-type mice using transgenic mice expressing green fluorescent protein (GFP) constitutively (driven by β-actin promoter) or selectively in endothelial cells (under Tie2 promoter activation) as donors. The presence of donor BMDCs in recipient mice was investigated in fat tissue vessels after DIO using in vivo and ex vivo fluorescence microscopy. We investigated the roles of VEGF receptors 1 and 2 (VEGFR1/VEGFR2) by inducing DIO in mice and treating them with blocking monoclonal antibodies. We found only marginal (less than 1%) incorporation of BMDCs in fat vessels during DIO. When angiogenesis was inhibited by blocking VEGFR2 in mice with DIO, treated mice had significantly lower body weights than control animals. In contrast, blocking VEGFR1 had no discernable effect on the weight gain during DIO. Conclusions/Significance: Formation of new vessels in fat tissues during DIO is largely due to angiogenesis rather than de novo vasculogenesis. Antiangiogenic treatment by blockade of VEGFR2 but not VEGFR1 may limit adipose tissue expansion

Characterization of water and wildlife strains as a subgroup of Campylobacter jejuni using DNA microarrays.

Campylobacter jejuni is the leading cause of human bacterial gastroenteritis worldwide, but source attribution of the organism is difficult. Previously, DNA microarrays were used to investigate isolate source, which suggested a non-livestock source of infection. In this study we analysed the genome content of 162 clinical, livestock and water and wildlife (WW) associated isolates combined with the previous study. Isolates were grouped by genotypes into nine clusters (C1 to C9). Multilocus sequence typing (MLST) data demonstrated that livestock associated clonal complexes dominated clusters C1-C6. The majority of WW isolates were present in the C9 cluster. Analysis of previously reported genomic variable regions demonstrated that these regions were linked to specific clusters. Two novel variable regions were identified. A six gene multiplex PCR (mPCR) assay, designed to effectively differentiated strains into clusters, was validated with 30 isolates. A further five WW isolates were tested by mPCR and were assigned to the C7-C9 group of clusters. The predictive mPCR test could be used to indicate if a clinical case has come from domesticated or WW sources. Our findings provide further evidence that WW C. jejuni subtypes show niche adaptation and may be important in causing human infection

Subcutaneous daratumumab and hyaluronidase-fihj in newly diagnosed or relapsed/refractory multiple myeloma.

Daratumumab, a human immunoglobulin G1 kappa monoclonal antibody that targets CD38, is currently approved as monotherapy and in varying combinations with approved anti-myeloma regimens in both newly diagnosed multiple myeloma and relapsed refractory multiple myeloma. Originally developed for intravenous administration, the subcutaneous formulation of daratumumab (daratumumab and hyaluronidase-fihj) was recently approved by the US Federal Drug Administration and European Commission in 2020. In clinical trials, compared with the intravenous formulation, subcutaneous daratumumab (Dara-SC) has significantly shorter administration time (median first dose 7 h versus 3-5 min, respectively), lower rates of infusion-related reactions (median first dose 50% versus less than 10%, respectively), and lower volume of infusion (median 500-1000 ml versus 15 ml, respectively). Otherwise, the pharmacokinetics, safety profile, and efficacy are comparable. This review summarizes the pivotal trials that led to the approval of Dara-SC, highlights important clinical considerations for the use of Dara-SC, and provides practical guidelines for the administration of Dara-SC in the clinic

Arf6 controls beta-amyloid production by regulating macropinocytosis of the Amyloid Precursor Protein to lysosomes

Alzheimer\u27s disease (AD) is characterized by the deposition of Beta-Amyloid (Aβ) peptides in the brain. Aβ peptides are generated by cleavage of the Amyloid Precursor Protein (APP) by the β - and γ - secretase enzymes. Although this process is tightly linked to the internalization of cell surface APP, the compartments responsible are not well defined. We have found that APP can be rapidly internalized from the cell surface to lysosomes, bypassing early and late endosomes. Here we show by confocal microscopy and electron microscopy that this pathway is mediated by macropinocytosis. APP internalization is enhanced by antibody binding/crosslinking of APP suggesting that APP may function as a receptor. Furthermore, a dominant negative mutant of Arf6 blocks direct transport of APP to lysosomes, but does not affect classical endocytosis to endosomes. Arf6 expression increases through the hippocampus with the development of Alzheimer\u27s disease, being expressed mostly in the CA1 and CA2 regions in normal individuals but spreading through the CA3 and CA4 regions in individuals with pathologically diagnosed AD. Disruption of lysosomal transport of APP reduces both Aβ40 and Aβ42 production by more than 30 %. Our findings suggest that the lysosome is an important site for Aβ production and that altering APP trafficking represents a viable strategy to reduce Aβ production

Longitudinal, 3D in vivo imaging of sebaceous glands by coherent anti-Stokes Raman scattering microscopy –normal function and response to cryotherapy

Sebaceous glands perform complex functions, and are centrally involved in the pathogenesis of acne vulgaris. Current techniques for studying sebaceous glands are mostly static in nature, whereas the gland’s main function – excretion of sebum via the holocrine mechanism – can only be evaluated over time. We present a longitudinal, real-time alternative – the in vivo, label-free imaging of sebaceous glands using Coherent Anti-Stokes Raman Scattering (CARS) microscopy, which is used to selectively visualize lipids. In mouse ears, CARS microscopy revealed dynamic changes in sebaceous glands during the holocrine secretion process, as well as in response to damage to the glands caused by cooling. Detailed gland structure, plus the active migration of individual sebocytes and cohorts of sebocytes were measured. Cooling produced characteristic changes in sebocyte structure and migration. This study demonstrates that CARS microscopy is a promising tool for studying the sebaceous gland and its associated disorders in three-dimensions in vivo